Glycomimetic affinity-enrichment proteomics identifies partners for a clinically-utilized iminosugar

Abstract

Widescale evaluation of interacting partners for carbohydrates is an underexploited area. Probing of the ‘glyco-interactome’ has particular relevance given the lack of direct genetic control of glycoconjugate biosynthesis. Here we design, create and utilize a natural product-derived glycomimetic iminosugar probe in a Glycomimetic Affinity-enrichment Proteomics (glyco-AeP) strategy to elucidate key interactions directly from mammalian tissue. The binding partners discovered here and the associated genomic analysis implicate a subset of chaperone and junctional proteins as important in male fertility. Such repurposing of existing therapeutics thus creates direct routes to probing in vivo function. The success of this strategy suggests a general approach to discovering ‘carbohydrate-active’ partners in biology.

Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149

“…Immobilized glycomimetic molecules, such as N- butyldeoxynojirimycin, have been used in affinity-enrichment proteomics to identify iminosugar-interacting proteins from mammalian tissue ( 39 ). Affinity-enrichment proteomics was performed using N- 5-carboxypentyl-DNJ immobilized on agarose beads as a glycomimetic probe to identity DNJ-interacting phosphorylase candidates in the Euglena Peak II sample.…”

“…The protocol was modified from Ref. 39 ; N- 5-carboxypentyl-DNJ (50 μmol; Toronto Research Chemicals) was conjugated to resin (1 ml; Bio-Rad Affidex 102 resin) overnight in anhydrous methanol using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (10 mg; Bio-Rad) as a catalyst. The reaction was performed at room temperature with gentle agitation.…”

A chemical genetic screen reveals that iminosugar inhibitors of plant glucosylceramide synthase inhibit root growth in Arabidopsis and cereals

“…Recently the link between starch and cell wall metabolism in germinating barley has been dissected using the iminosugar 1,4-dideoxy-1,4-imino- l -arabinitol (LAB), an inhibitor of arabinoxylan-hydrolase enzymes 25 . Other potential targets for iminosugars in plants include the carbohydrate binding proteins involved in cellular regulation and glycolipid processing enzymes 4 , 26 .…”

Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O-GlcNAc Proteins by Mass Spectrometry

“…Glycosylation, one of the most abundant types of protein modification, is found in more than 50% of proteins in cells. − Among the many different types of protein glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) is a unique and essential type of glycosylation that dynamically cycles on and off serine and threonine residues in nucleocytoplasmic proteins, including transcription factors, RNA binding proteins, signaling kinase, and cytoskeletal proteins. − O-GlcNAc modification plays critical roles in numerous cellular functions, such as gene expression, circadian rhythms, signal transduction, cell cycle, and protein localization. − The extent of GlcNAcylation on nucleocytoplasmic proteins is highly sensitive to the concentrations of glucose and other nutrients surrounding cells and therefore it serves as a nutrition sensor in regulating multiple metabolic pathways. − Altered O-GlcNAcylation is correlated with many major chronic diseases and the cross-talk between O-GlcNAcylation and phosphorylation − has been studied to understand the occurrence and development of neurodegenerative diseases, diabetes, and cancer. ,, Abnormal O-GlcNAcylation is increasingly accepted as a general feature of many types of cancer, indicating its potential as a new hallmark of cancer. − Therefore, global mapping and in-depth study of protein O-GlcNAcylation is important for understanding its cellular function and may lead to the discovery of potential therapeutic targets for disease treatment.…”